Severe sepsis, a clinical syndrome in patients with infection, surgery, trauma, or other injury, is associated with high mortality and limited effective therapeutics. High mobility group B1 (HMGB1), an endogenous molecule released by macrophages and other immunocompetent cells, is a necessary and sufficient mediator of lethal sepsis. Anti-HMGB1 antibodies that neutralize the cytokine activity of HMGB1 confer significant protection against inflammation, organ damage, and lethality in diverse animal models including severe sepsis, arthritis, pancreatitis, burn injury, acute lung injury, cerebral ischemia, hemorrhagic shock, colitis, and other inflammation and injury syndromes. The central tenet of the HMGB1 paradigm is that it mediates biological functions ranging from activating macrophages to secrete cytokines, to stimulating neutrophil migration. At present there are several major unanswered questions in this field, including: how do post-translational modifications alter HMGB1 interactions with TLR4 and RAGE, its two best understood and pre-eminent receptors? What is the effect of the HMGB1-TLR4 complex on activating intracellular signal transduction that drives cytokine release? Do HMGB1-TLR4 and HMGB1-RAGE complex formation mediate unique biological responses that can be dissociated in the context of sepsis? These questions will be addressed in the following Aims: Specific Aim 1: To elucidate whether cysteine modifications influence interaction of HMGB1-RAGE. Specific Aim 2. To elucidate whether HMGB1-TLR4 activates TIRAP-MyD88 and TRAM-TRIF dependent signaling. Specific Aim 3. To determine the beneficial effects of HMGB1- TLR4 or HMGB1-RAGE interaction in attenuating disease severity in sepsis. We propose to utilize an innovative strategy based on surface plasmon resonance to study HMGB1 binding to TLR4 and RAGE, substitution of specific amino acids to modify binding and signaling, proximity ligation assays to study intracellular binding of HMGB1 to TLR4 and RAGE, wholly synthetic peptides based on post- translational modified HMGB1 to produce competitive antagonists, and knockout mice to study these significant mechanisms in the context of lethal sepsis.